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Introduction The brain is a highly metabolic organ requiring a consistent supply of oxygen and nutrients for normal function. Ensuring a sufficient oxygen and nutrition supply to the brain is [...], Oxidative stress plays an important role in aging-related neurodegeneration. This study used littermates of WT and Nox2-knockout (Nox2KO) mice plus endothelial cell-specific human Nox2 overexpression-transgenic (HuNox2Tg) mice to investigate Nox2-derived ROS in brain aging. Compared with young WT mice (3-4 months), aging WT mice (20-22 months) had obvious metabolic disorders and loss of locomotor activity. Aging WT brains had high levels of angiotensin II (Ang II) and ROS production; activation of ERK1/2, p53, and [gamma]H2AX; and losses of capillaries and neurons. However, these abnormalities were markedly reduced in aging Nox2KO brains. HuNox2Tg brains at middle age (11-12 months) already had high levels of ROS production and activation of stress signaling pathways similar to those found in aging WT brains. The mechanism of Ang II-induced endothelial Nox2 activation in capillary damage was examined using primary brain microvascular endothelial cells. The clinical significance of Nox2-derived ROS in aging-related loss of cerebral capillaries and neurons was investigated using postmortem midbrain tissues of young (25-38 years) and elderly (61-85 years) adults. In conclusion, Nox2 activation is an important mechanism in aging-related cerebral capillary rarefaction and reduced brain function, with the possibility of a key role for endothelial cells.

TGF-[beta]1 levels increase after vascular injury and promote vascular smooth muscle cell (VSMC) proliferation. We define a nonviral gene delivery system that targets [alpha]v[beta]3 and [alpha]5[beta]1 integrins that are expressed on proliferating VSMCs and strongly induced by TGF-[beta]1. A 15-amino acid RGDNP-containing peptide from American Pit Viper venom was linked to a Lys(16) peptide as vector (molossin vector) and complexed with Lipofectamine or fusogenic peptide for delivery of luciferase or [beta]-galactosidase reporter genes to primary cultures of human, rabbit, and rat VSMCs. Preincubation of VSMCs with TGF-[beta]1 for 24 h, but not with PDGF-BB, interferon-[gamma] TNF-[alpha], nor PMA, increased [alpha]v[beta]3 and [alpha]5[beta]1 expressions on VSMCs and enhanced gene delivery of molossin vector. Thus [beta]-galactosidase activity increased from 35 [+ or -] 5% (controls) to 75 [+ or -] 5% after TGF-[beta]1 treatment, and luciferase activity increased fourfold over control values. Potential use of this system in vessel bypass surgery was examined in an ex vivo rat aortic organ culture model after endothelial damage. Molossin vector system delivered [beta]-galactosidase to VSMCs in the vessel wall that remained for up to 12 days posttransfection. The molossin vector system, when combined with TGF-[beta]1, enhances gene delivery to proliferating VSMCs and might have clinical applications for certain vasculoproliferative diseases. integrin; peptide vector; molossin rector

Tumor necrosis factor alpha (TNF-α) receptor-associated factors (TRAFs) play important roles in TNF-α signaling by interacting with downstream signaling molecules, e.g., mitogen-activated protein kinases (MAPKs). However, TNF-α also signals through reactive oxygen species (ROS)-dependent pathways. The interrelationship between these pathways is unclear; however, a recent study suggested that TRAF4 could bind to the NADPH oxidase subunit p47phox. Here, we investigated the potential interaction between p47phox phosphorylation and TRAF4 binding and their relative roles in acute TNF-α signaling. Exposure of human microvascular endothelial cells (HMEC-1) to TNF-α (100 U/ml; 1 to 60 min) induced rapid (within 5 min) p47phox phosphorylation. This was paralleled by a 2.7- ± 0.5-fold increase in p47phox-TRAF4 association, membrane translocation of p47phox-TRAF4, a 2.3- ± 0.4-fold increase in p47phox-p22phox complex formation, and a 3.2- ± 0.2-fold increase in NADPH-dependent O2- production (all P < 0.05). TRAF4-p47phox binding was accompanied by a progressive increase in extracellular signal-regulated kinases 1 and 2 (ERK½) and p38MAPK activation, which was inhibited by an O2- scavenger, tiron. TRAF4 predominantly bound the phosphorylated form of p47phox, in a protein kinase C-dependent process. Knockdown of TRAF4 expression using siRNA had no effect on p47phox phosphorylation or binding to p22phox but inhibited TNF-α-induced ERK½ activation. In coronary microvascular EC from p47phox-/- mice, TNF-α-induced NADPH oxidase activation, ERK½ activation, and cell surface intercellular adhesion molecule 1 (ICAM-1) expression were all inhibited. Thus, both p47phox phosphorylation and TRAF4 are required for acute TNF-α signaling. The increased binding between p47phox and TRAF4 that occurs after p47phox phosphorylation could serve to spatially confine ROS generation from NADPH oxidase and subsequent MAPK activation and cell surface ICAM-1 expression in EC. [ABSTRACT FROM AUTHOR]

TGF-β1 levels increase after vascular injury and promote vascular smooth muscle cell (VSMC) proliferation. We define a nonviral gene delivery system that targets αvβ3 and α5β1 integrins that are expressed on proliferating VSMCs and strongly induced by TGF-β1. A 15-amino acid RGDNP-containing peptide from American Pit Viper venom was linked to a Lys(16) peptide as vector (molossin vector) and complexed with Lipofectamine or fusogenic peptide for delivery of luciferase or β-galactosidase reporter genes to primary cultures of human, rabbit, and rat VSMCs. Preincubation of VSMCs with TGF-β1 for 24 h, but not with PDGF-BB, interferon-γ, TNF-α, nor PMA, increased αvβ3 and α5β1 expressions on VSMCs and enhanced gene delivery of molossin vector. Thus β-galactosidase activity increased from 35 ± 5% (controls) to 75 ± 5% after TGF-β1 treatment, and luciferase activity increased fourfold over control values. Potential use of this system in vessel bypass surgery was examined in an ex vivo rat aortic organ culture model after endothelial damage. Molossin vector system delivered β-galactosidase to VSMCs in the vessel wall that remained for up to 12 days posttransfection. The molossin vector system, when combined with TGF-β1, enhances gene delivery to proliferating VSMCs and might have clinical applications for certain vasculoproliferative diseases. [ABSTRACT FROM AUTHOR]

The p47phox is a key regulatory subunit of Nox2-containing NADPH oxidase (Nox2) that by generating reactive oxygen species (ROS) plays an important role in Angiotensin II (AngII)-induced cardiac hypertrophy and heart failure. However, the signalling pathways of p47phox in the heart remains unclear. In this study, we used wild-type (WT) and p47phox knockout (KO) mice (C57BL/6, male, 7-month-old, n = 9) to investigate p47phox-dependent oxidant-signalling in AngII infusion (0.8 mg/kg/day, 14 days)-induced cardiac hypertrophy and cardiomyocyte apoptosis. AngII infusion resulted in remarkable high blood pressure and cardiac hypertrophy in WT mice. However, these AngII-induced pathological changes were significantly reduced in p47phox KO mice. In WT hearts, AngII infusion increased significantly the levels of superoxide production, the expressions of Nox subunits, the expression of PKCα and C-Src and the activation of ASK1 (apoptosis signal-regulating kinase 1), MKK3/6, ERK1/2, p38 MAPK and JNK signalling pathways together with an elevated expression of apoptotic markers, i.e., γH2AX and p53 in the cardiomyocytes. However, in the absence of p47phox, although PKCα expression was increased in the hearts after AngII infusion, there was no significant activation of ASK1, MKK3/6 and MAPKs signalling pathways and no increase in apoptosis biomarker expression in cardiomyocytes. In conclusion, p47phox-dependent redox-signalling through ASK1, MKK3/6 and MAPKs plays a crucial role in AngII-induced cardiac hypertrophy and cardiomyocyte apoptosis. [ABSTRACT FROM AUTHOR]

Objective: To define the mechanism of p47(phox) phosphorylation in regulating endothelial cell response to tumor necrosis factor-α (TNFα) stimulation., Methods and Results: We replaced 11 serines (303-4, 310, 315, 320, 328, 345, 348, 359, 370, and 379) with alanines and investigated their effects on TNFα (100 U/mL, 30 minutes)-induced acute O(2)(.-) production and mitogen-activated protein kinase phosphorylation in endothelial cells. Seven constructs, S303-4A (double), S310A, S315A, S328A, S345A, S370A, and S379A, significantly reduced the O(2)(.-) production, and 4 of them (S328A, S345A, S370A, and S379A) also inhibited TNFα-induced extracellular-signal-regulated kinase (ERK) 1/2 phosphorylation. Blocking the phosphorylation of S303-4 and S379 inhibited most effectively TNFα-induced O(2)(.-) production. However, phosphorylation of S303-4 was not required for TNFα-induced p47(phox) membrane translocation and binding to TNF receptor-associated factor 4, ERK1/2 activation, and subsequent vascular cell adhesion molecule-1 expression. Knockout of p47(phox) or knockdown of TNF receptor-associated factor 4 using siRNA abolished TNFα-induced ERK1/2 phosphorylation, and inhibition of ERK1/2 activation significantly reduced the TNFα-induced vascular cell adhesion molecule-1 expression., Conclusions: Phosphorylation of p47(phox) at different serine sites plays distinct roles in endothelial cell response to TNFα stimulation. Double serine (S303-4) phosphorylation is crucial for acute O(2)(.-) production, but is not involved in TNFα signaling through TNF receptor-associated factor 4 and ERK1/2. p47(phox) requires serine phosphorylation at distinct sites to support specific signaling events in response to TNFα.

Background: NADPH oxidase is a major source of vascular superoxide (O2-) production and is implicated in angiotensin II (Ang II)-induced oxidant stress. The p47phox subunit plays an important role in Ang II-induced oxidase activation, but its role in basal oxidase activity and vascular function is unclear., Methods and Results: Aortae from p47phox-/- and matched wild-type (WT) mice (n=9/group) were incubated ex vivo with or without Ang II (200 nmol/L, 30 minutes) and then examined for (1) NADPH-dependent O2- production, (2) endothelium-dependent and -independent vascular relaxation, and (3) activation of mitogen-activated protein kinases (MAPKs). In the absence of Ang II, p47phox-/- vessels had slightly but significantly higher (1.3+/-0.1-fold; P<0.05) NADPH-dependent O2- production than WT; impaired relaxation to acetylcholine (maximum 54+/-4% versus 80+/-3%; P<0.05), which was normalized to WT levels by the O2- scavenger tiron or by Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin pentachloride, and increased basal phosphorylation of ERK1/2, p38MAPK, and JNK compared with WT. In WT aortae, Ang II increased NADPH-dependent O2- production (2.5+/-0.5-fold; P<0.05), impaired relaxation to acetylcholine (maximum 60+/-6% versus 80+/-3%; P<0.05), and increased ERK1/2, p38MAPK, and JNK phosphorylation (P<0.05). In contrast, Ang II failed to increase O2- production, impair acetylcholine responses, or increase MAPK activation in p47phox-/- aortae., Conclusions: p47phox plays a complex dual role in the vasculature. It inhibits basal NADPH oxidase activity but is critical for Ang II-induced vascular dysfunction via activation of NADPH oxidase.